Loss of CDH16 expression is a strong independent predictor for lymph node metastasis in Middle Eastern papillary thyroid cancer

Papillary Thyroid Cancer (PTC) is the most common type of thyroid cancer. The membrane-associated glycoprotein cadherin-16 (CDH16) plays a significant role in the embryonal development of thyroid follicles and cell adhesion. Previous studies have indicated a substantial downregulation of CDH16 in PTC. However, its role in Middle Eastern PTC has not been elucidated. We analyzed a tissue microarray comprising 1606 PTC and 240 normal thyroid tissues using immunohistochemistry to assess CDH16 expression and determine its clinico-pathological associations. We also conducted BRAF and TERT mutations analyses through Sanger sequencing. Disease-free survival (DFS) was assessed using Kaplan–Meier curves. CDH16 immunostaining was seen in 100% of normal thyroid tissues but only in 9.4% of PTC tissues (p < 0.0001). The loss of CDH16 expression was associated with aggressive PTC characteristics including bilaterality, multifocality, extrathyroidal extension, tall cell variant, lymph node metastasis (LNM) and distant metastasis. Additionally a correlation between loss of CDH16 expression and BRAF and TERT mutations was identified. Intriguingly, upon conducting multivariate logistic regression analysis, CDH16 was determined to be an independent predictor for LNM (Odds ratio = 2.46; 95% confidence interval = 1.60–3.79; p < 0.0001). Furthermore, CDH16 loss was associated with a shorter DFS (p = 0.0015). However, when we further subdivided CDH16 negative patients based on the co-existence of TERT and/or BRAF mutations, we found that patients with both CDH16 negative expression and TERT mutation exhibited the shortest DFS (p < 0.0001). In conclusion, our results suggest that CDH16 protein expression could serve as a valuable diagnostic tool for PTC. Furthermore, these findings demonstrate that the loss of CDH16 expression is an independent predictor of LNM and may contribute to the aggressiveness of PTC. Therefore, downregulation of CDH16 in PTC might be a potential target for designing novel therapeutic strategies to treat PTC.

recurrences and providing clinicians with valuable guidance when making therapeutic and follow-up decisions for PTC patients.
Cadherin-16 (CDH16) belongs to the Cadherin superfamily, which encompasses various calcium-dependent membrane-associated glycoproteins 14 .CDH16 serves a pivotal role in maintaining cell adhesion, embryonal development and cell growth 14,15 .Its crucial involvement in mediating cell-cell adhesion has led to several studies highlighting a strong association between abnormal CDH expression and tumor progression and metastasis [16][17][18] .A recent study has shed light on the prognostic significance of CDH16 in renal cell carcinoma, where a reduction in CDH16 expression was identified as a potent predictor of poor prognosis 19 .Additionally, CDH16 has been implicated in the development and differentiation of thyroid gland during embryogenesis 20,21 .Interestingly, our recent study, conducted in a smaller cohort encompassing multiple organ sites revealed a lower frequency of CDH16 expression in PTC compared to normal thyroid, follicular adenomas and follicular carcinomas 22 .RNA expression studies have also suggested a significant downregulation of CDH16 in PTC in comparison to thyroiditis 23 .Utilizing The Cancer Genome Atlas (TCGA) cohort and bioinformatics analysis, they showed an association between CDH16 expression and unfavorable clinico-pathological features in TC.Moreover, a recent investigation elucidated the mechanistic role of CDH16 in TC, where CDH16 overexpression inhibited cell proliferation and migration, while inducing apoptosis, affirming its role as tumor suppressor 24 .Furthermore, by analyzing a small cohort of 35 PTC patients, they demonstrated a significant correlation between low expression of CDH16 and tumor size, stage and LNM.
Despite these intriguing prior findings, research on the immunohistochemical expression of CDH16 has predominantly focused on renal cell carcinomas [25][26][27][28][29][30][31][32] , with only one recent study in PTC 22 .Additionally, publicly accessible RNA databases have indicated CDH16 expression in cervical, endometrial and ovarian cancers [33][34][35] .Nevertheless, comprehensive studies involving substantial cohorts of PTC patients remain scarce.As a result, this study was specifically designed to investigate the expression of CDH16 and its potential utility as a prognostic and diagnostic marker.We conducted our study using a cohort comprising 240 normal thyroid tissues and 1606 PTC samples by immunohistochemistry. Furthermore, we delved into the clinico-pathological and molecular correlations associated with CDH16 expression in Middle Eastern PTC cases.
We further analyzed the CDH16 expression in pediatric/adolescent patients (≤ 18 years) in our cohort.Patients aged ≤ 18 years constituted 5.6% (90/1606) of the total cases.CDH16 expression was noted in 6.7% (6/90) of pediatric/adolescent cases.However, the difference in CDH16 expression between pediatric/adolescent and adult PTC was not statistically significant (p = 0.4581) (Table 1).

Disease-free survival
Loss of CDH16 expression was found to be associated with significantly shorter disease-free survival (DFS) (p = 0.0015, Fig. 2A).Given the significant association of BRAF and TERT mutations with CDH16, we further www.nature.com/scientificreports/analyzed the effect of these mutations on CDH16 loss of expression in predicting DFS.We divided the patients with CDH16 loss into four categories: CDH16 negative alone, CDH16 negative + BRAF mutant, CDH16 negative + TERT mutant and CDH16 negative + BRAF mutant + TERT mutant.Interestingly, we found that CDH16 negative + TERT mutant patients had the worst DFS among the four groups (p < 0.0001, Fig. 2B).

Discussion
In this study, we observed a significant downregulation of CDH16 expression in PTC when compared to normal thyroid tissue.This loss of CDH16 was found to be associated with aggressive clinico-pathological characteristics and shorter DFS.The multivariate logistic regression analysis further confirmed that CDH16 stands as an independent predictor for LNM.Interestingly, when we sub-grouped CDH16 negative patients based on the presence of co-existing TERT and/or BRAF mutations, we discovered that patients with co-existing CDH16 negative expression and TERT mutation exhibited the shortest DFS.
In a recent study conducted by Lennartz et al. 22 , which analyzed over 10,000 tumors spanning more than 100 entities, CDH16 positivity was identified in 100% of normal thyroid tissues, 86% of 94 follicular adenomas, 60% of 67 follicular carcinomas and only 6.6% of 212 papillary thyroid carcinomas.In this study, a collection of tissue microarrays containing 1846 samples from 1606 PTC and 240 normal thyroid tissues revealed CDH16 membranous immunostaining in 100% of normal thyroid tissues, but only 9.4% of PTCs.Our finding regarding the low CDH16 positivity rate in PTC is in concordance with the results of Lennartz et al. 22 .However, it is worth noting that another study by Yang et al. 24 reported CDH16 positivity in 51.4% of PTCs, albeit in a much smaller sample size of 35 cases.The presence of CHD16 expression in 100% of normal thyroid tissues is consistent with previous research, indicating that CDH16 expression is associated with the fully differentiated state of thyroid cells and plays a role in thyroid follicular polarity 20,21,36 .The striking absence of CDH16 expression in over 90% of PTC cases suggests a relevant clinical practical utility of using CDH16 immunostaining as a useful diagnostic tool for the identification of these tumors.
Our analysis, encompassing a substantial cohort of over 1600 PTCs, enabled us to investigate in depth the correlation between the loss of CDH16 expression and various clinico-pathological and molecular parameters.Although the loss of CDH16 expression was unrelated to age, gender, tumor size and disease stage, it exhibited strong associations with several other aggressive features like tall cell variant, extrathyroidal extension, multifocality and bilateral tumors.Most notably, we observed a significant association between the absence of CDH16 and lymph node metastasis (LNM), as well as distant metastasis.Subsequent logistic regression analyses showed that downregulated expression of CDH16 was an independent predictor of LNM.Based on the identified role of CDH16 as cell adhesion molecule that preserves tissue integrity and inhibits cell migration and invasion 24 , the increased level of aggressiveness in PTC with the loss of CDH16 expression might be driven by lower degree of cells organization and higher tumor cell ability of motility and migration.Our findings are in concordance with results from previous studies that have described reduced CDH16 expression in association with aggressive PTC phenotype 23,24 .Both Li et al. 23 (mRNA expression) and Yang et al. 24 (protein expression) found a significant association between the downregulation of CDH16 and LNM, with Li et al. further demonstrating that reduced expression of CDH16 was an independent predictor of LNM, consistent with our study.These findings suggest that loss of CDH16 could be associated with PTC progression.We were particularly intrigued by the association of CDH16 expression loss and BRAF as well as TERT mutation.Previous studies have shown an interaction between CDH16 and other cancer genes 23,37 .In this cohort, lack of CDH16 expression was significantly associated with poor DFS in univariate analysis using the Kaplan-Meier curve.Since both BRAF and TERT mutations are known to be aggressive markers for PTC [38][39][40][41][42] and both have been shown to be associated with poor DFS, we asked whether this survival effect of CDH16 loss of expression was affected by coexisting mutations in TERT and BRAF gene mutations.Specifically, we compared the DFS of PTC patients with coexisting TERT and BRAF mutations to those with a lack of CDH16 and found that PTC patients with coexisting lack of CDH16 and the presence of TERT mutation had the worst DFS.This is not surprising, given the clinical and functional role of TERT in PTC, which we and others have shown previously 38,43,44 .TERT is known to promote cancer progression, leading to the loss of epithelial cell adhesion molecule, E-cadherin and induction of several mesenchymal markers such as N-cadherin and vimentin 38,45,46 .The synergistic effect of TERT and CDH16 on cellular adhesion and cancer progression may explain the worse DFS when TERT mutation and CDH16 loss coexist.
Despite these interesting findings and the large cohort included in this study, this research still has some limitations.First, this is retrospective single institute cohort from Middle Eastern ethnicity where selection bias cannot be excluded.Second, the functional mechanism between CDH16 and other genes are mainly theoretical and should be further investigated and validated.Thirdly, the results of our findings cannot be generalized to the global population and hence, future large scale studies in other ethnicities is recommended.
In conclusion, our study has revealed that CDH16 protein expression is massively downregulated in Middle Eastern PTC when compared to normal tissue, indicating its potential as a valuable diagnostic tool for identification of these tumors.Furthermore, we have provided crucial clinical evidence that lack of CDH16 expression promotes PTC aggressiveness and serves as an independent predictor for LNM in PTC patients.Patients with both CDH16 expression loss and TERT mutation exhibited the worst DFS.This study underscores the potential of targeting CDH16 expression in PTC as a promising avenue for the development of therapeutic strategies to treat PTC.

Patient selection and clinico-pathological data
One thousand six-hundred and six PTC patients diagnosed between 1988 and 2020 at King Faisal Specialist Hospital and Research Centre (Riyadh, Saudi Arabia) were included in the study.Cases were identified based on clinical history followed by fine needle aspiration cytology for confirmation.Baseline clinico-pathological data were collected from case records and has been summarized in Table 1.Staging of PTC was performed using the eighth edition of American Joint Committee on Cancer (AJCC) staging system 47

BRAF and TERT mutation analysis
BRAF and TERT mutation status was assessed in our laboratory by utilizing Sanger sequencing technology and has been published by us previously 38,48,49 .BRAF mutation analysis was performed in 1574 cases and TERT mutation analysis was done in 1320 cases.

Tissue microarray (TMA) construction and immunohistochemistry (IHC) analysis
Tissue microarray (TMA) format was utilized for immunohistochemical analysis of the PTC samples.TMA was constructed as previously described 50 .Briefly, modified semiautomatic robotic precision instrument (Beecher Instruments, Woodland, WI) was used to punch tissue cylinders with a diameter of 0.6 mm from representative tumor area of the donor tissue block and brought into the recipient paraffin block.Two 0.6-mm cores of PTC were arrayed from each case.Tissue microarray slides were processed and stained manually as described previously 51 .Primary antibody against CDH16 (monoclonal Recombinant rabbit, MSVA-516R, MS Validated Antibodies, Hamburg, Germany) was used at a dilution of 1:200 (pH 9).The Dako Envision Plus System kit was used as the secondary detection system with 3, 30-diaminobenzidine as chromogen.All slides were counter stained with hematoxylin, dehydrated, cleared and mounted.Negative controls included omission of the primary antibody.Normal tissues of different organ system were also included in the TMA to serve as control.Only fresh cut slides were stained simultaneously to minimize the influence of slide aging and maximize reproducibility of the experiment.
Staining was scored as described previously 19 .Briefly, the percentage of CDH16 positive tumor cells was estimated and the staining intensity was semi-quantitatively assessed (0, 1+, 2+ and 3+).For statistical analyses, staining results were categorized into two groups: Negative-no staining at all; Positive-staining of any intensity.

Follow-up and study endpoint
Patients were regularly followed by both physical examinations and imaging studies to identify tumor persistence/ recurrence.The median follow-up was 7.5 years (range 1.0-30.2years).The study end-point was DFS.Patients were grouped according to disease status, with patients considered to be disease-free in the absence of clinical, biochemical (unstimulated serum thyroglobulin (Tg) levels of < 0.2 µg/L or stimulated Tg levels of < 1 µg/L in the absence of interfering thyroglobulin antibodies (TgAb)) or radiological evidence of disease persistence or recurrence.In contrast, active disease was defined by the presence of unstimulated serum Tg levels ≥ 0.2 µg/L or stimulated Tg levels ≥ 1 µg/L; a rising or denovo appearance of TgAb; or abnormal findings on radio-imaging.

Statistical analysis
The associations between clinico-pathological variables and CDH16 protein expression was performed using contingency table analysis and Chi square tests.Mantel-Cox log-rank test was used to evaluate DFS.Survival curves were generated using the Kaplan-Meier method.Logistic regression analysis was used for univariate and multivariate analysis.Two-sided tests were used for statistical analyses with a limit of significance defined as p value < 0.05.Data analyses was performed using the JMP14.0(SAS Institute, Inc., Cary, NC) software package.

Figure 1 .
Figure 1.Examples of CDH16 immunostaining in normal thyroid and papillary Thyroid cancer (PTC).(A) Representative section of normal thyroid tissue showing strong positive membrane expression of CDH16 in the follicular cells.(B) PTC tissue showing positive staining for CDH16 and (C) another PTC tissue showing negative staining for CDH16 with adjacent normal tissue showing positive expression.(20 X/0.70 objective on an Olympus BX 51 microscope (Olympus America Inc, Center Valley, PA, USA)).

Figure 2 .
Figure 2. Disease-free survival (DFS).(A) Kaplan Meier survival plot showing statistically significant shorter DFS in CDH16 negative cases compared to CDH16 positive (p = 0.0015) (B) Kaplan Meier survival plot showing statistically significant shorter DFS in patients with co-existing CDH16 negative and TERT mutation (p < 0.0001), compared to other sub-groups.

Table 1 .
Clinico-pathological associations of CDH16 expression in papillary thyroid cancer.Statistical analyses were performed only on available data and unknown cases were excluded.